High level recombinant protein production using conditional helper-free adenovirus vector

Abstract

The present invention relates to methods and compositions involving an improved recombinant transfer vector for introducing a DNA sequence, encoding a recombinant protein, into an adenovirus genome in generating recombinant adenovirus. Recombinant protein production, in cells infected with the recombinant adenovirus, can approach levels as high as 15-20% of total cellular proteins. The improved transfer vector includes an expression cassette comprising sequentially a transcription promoter, a high efficiency leader, at least one splicing signal, an enhancer-like sequence, a cloning site and a plurality of polyadenylation sites.

We claim: 
     
       1. An adenovirus transfer vector for introducing a DNA sequence, encoding a recombinant protein, into an adenovirus genome in generating a helper-free adenovirus recombinant, said transfer vector: (a) contains an expression cassette comprising in sequential order, a first enhancer-like sequence as shown in SEQ ID NO:b 2, a transcriptional promoter, a high efficiency leader, at least one splicing signal, a second enhancer-like sequence as shown in SEQ ID NO:1, a cloning site and a plurality of polyadenylation sites; and   (b) provides a means for generating a helper-free adenovirus recombinant, wherein expression of recombinant protein by a mammalian cell infected with the helper-free adenovirus recombinant is at a level of about 10% to about 25% of total cellular proteins.   
     
     
       2. A helper-free adenovirus recombinant made by homologous recombination between the transfer vector of claim 1 containing a DNA sequence encoding a protein, and adenovirus genomic DNA. 
     
     
       3. The transfer vector of claim 1, wherein said transfer vector is pAdBM5, having ATCC Designation 75704, and wherein the first enhancer-like sequence comprises a BK virus enhancer sequence, the transcriptional promoter comprises an adenovirus MLP, the high efficiency leader comprises an adenovirus tripartite leader, and the second enhancer-like sequence comprises adenovirus binding sites R1, R2, and R3. 
     
     
       4. A helper-free adenovirus recombinant made by homologous recombination between the transfer vector of claim 3 containing a DNA sequence encoding a protein, and adenovirus genomic DNA.